Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

AbstractHere we introduce RNA-CLAMP, a technology which enables site-specific and enzymatic cross-linking (clamping) of two selected stem loops within an RNA of interest. Intramolecular clamping of the RNA can disrupt normal RNA function, whereas subsequent photo-cleavage of the crosslinker restores activity. We applied the RNA-CLAMP technique to the single guide RNA of the CRISPR-Cas9 gene editing system. By clamping two stem loops of the single-guide RNA (sgRNA) with a photo-cleavable cross-linker, gene editing was completely silenced. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Furthermore, by designing two photo-cleavable linkers which are responsive to different wavelength of lights, we achieved multiplexed photo-activation of gene editing in mammalian cells. Notably, although the Cas9-sgRNA RNP is not capable of DNA cleavage activity upon clamping, it maintained the capability to bind to the target DNA. The RNA-CLAMP enabled photo-activated CRISPR-Cas9 gene editing platform offers clean background, free choice of activation wavelength and multiplexing capability. We believe that this technology to precisely and rapidly control gene editing will serve as a versatile tool in the future development of stimuli responsive gene editing technologies. Beyond gene editing, RNA-CLAMP provides a site-specific tool for manipulating the internal structure of functional RNAs.

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RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

10.1101/848309 ◽

2019 ◽

Cited By ~ 1

Author(s):

Adrian Kovač ◽

Csaba Miskey ◽

Michael Menzel ◽

Esther Grueso ◽

Andreas Gogol-Döring ◽

...

Keyword(s):

Genome Engineering ◽

Sleeping Beauty ◽

Specific Gene ◽

Guide Rna ◽

Gene Insertion ◽

Genome Wide ◽

Asymmetric Pattern ◽

Integration Sites ◽

Genomic Regions ◽

Selection Of

ABSTRACTTwo different approaches of genomic modification are currently used for genome engineering and gene therapy: integrating vectors, which can efficiently integrate large transgenes but are unspecific with respect to their integration sites, and nuclease-based approaches, which are highly specific but not efficient at integrating large genetic cargoes. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, occurred in a relatively narrow, ∼200 bp window around the targeted sites and displayed an asymmetric pattern with a bias towards sites that are downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for efficient and specific gene insertion for precision genetic engineering.

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Site-specific Gene Insertion Mediated by a Cre-loxP-carrying Lentiviral Vector

Molecular Therapy ◽

10.1038/mt.2010.150 ◽

2010 ◽

Vol 18 (10) ◽

pp. 1814-1821 ◽

Cited By ~ 16

Author(s):

Gilles Michel ◽

Yin Yu ◽

Tammy Chang ◽

Jiing-Kuan Yee

Keyword(s):

Lentiviral Vector ◽

Specific Gene ◽

Site Specific ◽

Gene Insertion

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Footprint-Free, Site-Specific Gene Editing

Genetic Engineering & Biotechnology News ◽

10.1089/gen.33.5.10 ◽

2013 ◽

Vol 33 (5) ◽

pp. 18-19

Author(s):

Carlisle Landel

Keyword(s):

Gene Editing ◽

Specific Gene ◽

Site Specific

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414. Design of an HIV Vector for Site-Specific Gene Insertion

Molecular Therapy ◽

10.1016/j.ymthe.2004.06.429 ◽

2004 ◽

Vol 9 ◽

pp. S159

Keyword(s):

Specific Gene ◽

Site Specific ◽

Gene Insertion

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Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502370112 ◽

2015 ◽

Vol 112 (13) ◽

pp. 4038-4043 ◽

Cited By ~ 94

Author(s):

Sanjay Basu ◽

Azadeh Aryan ◽

Justin M. Overcash ◽

Glady Hazitha Samuel ◽

Michelle A. E. Anderson ◽

...

Keyword(s):

Aedes Aegypti ◽

Specific Gene ◽

Gene Drive ◽

Transcription Activator ◽

End Joining ◽

Site Specific ◽

Gene Insertion ◽

Guide Rnas ◽

Essential Components ◽

Drive Systems

Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24–90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2–3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

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